Method for growing tissue specimens in vitro

ABSTRACT

An apparatus and method for growing tissue specimens in vitro wherein the apparatus includes an expandable membrane for receiving a tissue specimen thereon, a mechanism for expanding the membrane and the tissue specimen, and a controller for controlling the expanding mechanism. The controller is operative in accordance with the method for applying an activity pattern to the membrane and a tissue specimen thereon which includes simultaneous continuous stretch activity and repetitive stretch and release activity. The continuous stretch activity and the repetitive stretch and release activity simulate the types of activity to which cells are exposed in vivo due to growth and movement, respectively, and they cause the cells of tissue specimens grown in the apparatus in accordance with the method to develop as three-dimensional structures similar to those grown in vivo.

BACKGROUND AND SUMMARY OF THE INVENTION

The instant invention relates to tissue cultures and more particularlyto an in vitro method and apparatus for growing tissue cells underconditions which more closely resemble cells grown in vivo.

Experiments wherein tissue cells are grown in in vitro tissue cultureshave produced significant amounts of data relating to tissue developmentover the past 70 years. However, it has generally been found that thecells of specimens which are grown in static, in vitro environmentsdevelop as randomly oriented embryonic cells which lack thethree-dimensional adult characteristics of cells grown in vivo.Recently, however, it has been found that by applying stimulationconsisting of gradual stretching forces to tissue specimens grown in invitro tissue cultures, the tissue cells thereof tend to align with thestretching forces and they therefore exhibit somewhat more maturecharacteristics. However, it has been found that even the cells ofspecimens which are exposed to stimulation consisting of gradualstretching forces fail to develop into adult-like, three-dimensionalstructures which are similar to those grown in vivo. It has alsorecently been found that by applying stimulation consisting ofrepetitive or reciprocating stretch and release activity to tissuespecimens grown in in vitro tissue cultures, the cells thereof tend toalign in perpendicular relation to the direction of the stretch andrelease activity, and still fail to develop into three-dimensionalstructures similar to those grown in vivo.

It has now been found that when in vitro culture cells are stimulated byapplying a mechanical activity pattern thereto which more closelyresembles the type of stimulation pattern which naturally occurs invivo, it is possible to grow the cells of a tissue specimen intostructures which more closely resemble tissue cells grown in vivo.Specifically, it has been found that by simultaneously applying bothcontinuous, gradual stretch activity and periodic reciprocal stretch andrelease activity to a tissue specimen grown in an in vitro culture, itis possible to develop the cells of the tissue specimen into wellformed, three-dimensional structures which are similar to those grown invivo. In this connection, by continuously stretching a tissue specimen,the tissue specimen is subjected to the type of mechanical stimulationwhich normally results from animal growth in vivo; and by periodicallyrepetitively stretching and releasing the tissue specimen, the tissuespecimen is subjected to the type of stimulation to which in vivo tissuecells are exposed during animal movements.

In accordance with the above, the method of the subject inventioncomprises the steps of supporting a tissue specimen in the grossmorphology found in vivo (aligned or unaligned as in vivo) with anexpandable membrane so that the tissue specimen is expandable therewith,maintaining the tissue specimen in an extracellular matrix, such as acollagen matrix, maintaining the tissue specimen in a life supportenvironment, periodically supplying fresh nutrients to the tissuespecimen, and gradually stretching the membrane by at leastapproximately 0.5% over a 24 hr period while periodically reciprocallystretching and releasing the membrane by at least approximately 0.01%for a total of at least 60 seconds during the course of the same 24 hrperiod. The tissue specimen is preferably stretched and released bybetween 0.02% and 60% during the stretch and release activity periods,and the stretch and release activity periods preferably last at leastapproximately 5 seconds. The stretch and release activity periods arepreferably separated by rest periods of at least approximately 5seconds, and the tissue specimen is preferably gradually andcontinuously stretched at a rate of between 0.005 mm/hr and 1.0 mm/hr.The membrane preferably has a pair of spaced, upwardly extending supportwalls thereon which are made of a biocompatible material and haveopposed support surfaces thereon, and the tissue specimen is preferablyallowed to attach to the support surfaces of the support walls, and itis stretched by moving the support walls apart.

When the method of the subject invention is applied to a tissue specimencomprising isolated cells requiring alignment, the tissue specimen ispreferably applied to a resiliently expandable membrane which is adaptedto permit attachment of the tissue specimen thereto, such as bypreapplying a collagen coating thereto. Thereafter, the method iscarried out by maintaining the tissue specimen in a life supportenvironment, periodically feeding the tissue specimen, and maintainingthe cells of the tissue specimen in an extracellular matrix sufficientto permit stretching of the tissue specimen without causing damagethereto. Then, while maintaining these conditions, the membrane isgradually stretched over a prolonged period of time until the cells aresubstantially aligned in the direction of stretch, and thereafter themembrane is gradually stretched by at least approximately 0.5% over thecourse of a 24 hr period while it is periodically stretched and releasedby at least approximately 0.01% for a total of at least 60 secondsduring the course of the same 24 hr period. The tissue specimen ispreferably stretched and released by between 0.02% and 60% during thestretch and release activity periods, and it is preferably graduallystretched at a rate of between 0.005 mm/hr and 1.0 mm/hr. The tissuespecimen is preferably repetitively stretched and released duringactivity periods of at least 5 seconds in duration, and the activityperiods are preferably separated by rest periods of at least fiveseconds in duration. The membrane preferably has a pair of spaced,upwardly extending support walls thereon, and the support walls arepreferably made of a biocompatible material and they have opposedsupport surfaces thereon. The support walls are preferably made ofstainless steel screening so that the support surfaces thereof havesufficiently roughened textures to permit attachment of the cellsthereto. In this regard, the tissue specimen is attached or allowed toattach to the support surfaces of the support walls, and thereafter thetissue specimen is mechanically stimulated by stretching the membrane tomove the support walls apart in accordance with the method.

The apparatus of the instant invention which is operative for performingthe above method comprises a resiliently expandable support means whichis operative for supporting a tissue specimen and adapted as needed topermit attachment of the tissue specimen thereto. The apparatus furthercomprises means for expanding the support means to stretch the tissuespecimen and control means for controlling the means for expanding thesupport means to gradually and substantially continuously expand thesupport means over a prolonged period of time and to repetitively expandor stretch and release the support means over the same period of time.The control means is preferably adapted to control the means forexpanding the support means to effect stretching activity in accordancewith the method as hereinabove set forth. In a first embodiment of theapparatus, the support means comprises a substantially circular,resiliently flexible membrane having an upwardly facing side and adownwardly facing side, and the tissue specimen is applied to theupwardly facing side of the membrane. In this embodiment, the mechanicalmanipulating means is engageable with the downwardly facing side of themembrane to deform the membrane upwardly. The support means preferablyfurther comprises a substantially circular support ring on the membranewhich extends upwardly from the membrane and has an inwardly facingsupport surface thereon. The support ring is made of a biocompatiblematerial, such as stainless steel screening, and it has a sufficientlyroughened surface texture to permit attachment of the tissue specimenthereto. In a second embodiment of the apparatus, the support meanscomprises a resiliently flexible membrane having opposite first andsecond ends and having an upwardly facing side and a downwardly facingside, and the mechanical manipulating means is operative for stretchingthe cells by separating the first and second ends. In this embodiment,the support means preferably further comprises first and second spacedsupport walls which extend upwardly from the upwardly facing side of themembrane and have opposed support surfaces thereon. The support wallsare also preferably made of a biocompatible screening material, such asstaninless steel screening, so that the support surfaces havesufficiently roughened textures to permit attachment of the tissuespecimen thereto.

Accordingly, it is a primary object of the instant invention to providean effective method of growing a tissue specimen in vitro so that thetissue specimen more closely resembles tissue which is grown in vivo.

Another object of the instant invention is to provide a method ofmechanically stimulating the growth of a tissue specimen by graduallyand substantially continuously stretching the tissue specimen over aprolonged period of time while simultaneously repetitively stretchingand releasing the tissue specimen.

Another object of the instant invention is to provide an apparatus formechanically stimulating the growth of a tissue specimen.

An even further object of the instant invention is to provide aneffective apparatus for mechanically stimulating the growth of a tissuespecimen by simultaneously gradually stretching the tissue specimen andperiodically repetitively stretching and releasing the tissue specimen.

Other objects, features and advantages of the invention shall becomeapparent as the description thereof proceeds when considered inconnection with the accompanying illustrative drawings.

DESCRIPTION OF THE DRAWINGS

In the drawings which illustrate the best mode presently contemplatedfor carrying out the present invention:

FIG. 1 is a fragmentary perspective view of a first embodiment of theapparatus of the instant invention;

FIG. 2 is a sectional view taken along line 2--2 in FIG. 1;

FIG. 3 is an enlarged, fragmentary, exploded perspective view of thecell assembly of the apparatus illustrated in FIG. 1;

FIG. 4 is an enlarged sectional view of the well assembly with tissuespecimens in the wells;

FIG. 5 is a graph illustrating a typical activity pattern applied by themethod;

FIG. 6 is a perspective view of a second embodiment of the apparatus;

FIG. 7 is an end elevational view thereof;

FIG. 8 is an exploded fragmentary perspective view of the well assemblythereof; and

FIG. 9 is an enlarged sectional view taken along line 9--9 in FIG. 8with tissue specimens in the wells.

DESCRIPTION OF THE INVENTION

Referring now to the drawings, a first embodiment of the apparatus forgrowing tissue specimens of the instant invention is illustrated inFIGS. 1-4 and generally indicated at 10 in FIGS. 1 and 2. The apparatus10 comprises a well assembly generally indicated at 12 having aplurality of wells 13 therein, a stimulator pin assembly generallyindicated at 14, and a controller 16. For operation of the apparatus 10,living tissue cells are placed in the wells 13 in the well assembly 12,and the controller 16 is actuated to control the stimulator pin assembly14 to mechanically stimulate the tissue cells with an activity patternwhich includes both gradual, substantially continuous stretch activityand repetitive stretch and release activity.

The well plate assembly 12 comprises a bottom plate 18 having aplurality of spaced apertures therein, a membrane 20, a gasket 22, andan upper plate 24. The membrane 20 is preferably made of a medicalgrade, resiliently flexible, rubberized material, such as Silastic (DowCorning Corp. TM), and it preferably has a thickness of approximately0.01 in. The upper surface of the membrane 20 is preferably precoatedwith an extracellular matrix material, such as rat tail collagen, toadapt the membrane 20 to permit the attachment of tissue cells thereto.The gasket 22 has a plurality of spaced bores therein, and it isreceived on the membrane 20 and adhesively secured thereto with asilicone rubber sealant so that the bores in the gasket 22 aresubstantially aligned with the apertures in the bottom plate 18. Theupper plate 24 is preferably made of Teflon (DuPont TM), and it has aplurality of bores 26 therein which are substantially aligned with thebores in the gasket 22. The upper plate 24, the gasket 22, the membrane20, and the bottom plate 18 are maintained in assembled relation withbolts 28. Also included in the well assembly 12 is a plurality ofsupport rings 30 which are received in the wells 13, the rings 30 havingroughened inner support surfaces 32 thereon which have sufficientroughness to permit attachment of tissue cells thereto. The rings 30 arepreferably made of a biocompatible screening material, such as stainlesssteel screening, and they are formed so that they are frictionallyretained against the sidewalls of the wells 13. The support rings 30 arepreferably made of screening having between 4×4 meshes/in and 250×250meshes/in, a wire diameter of between 0.31 mm and 3.76 mm, and meshopenings of between 0.20 mm and 2.59 mm; and as herein embodied, theyhave 50×50 meshes/in, a wire diameter of 0.31 mm, and mesh openings of0.20 mm. The rings 30 are received in the wells 13 so that they arepositioned on the membrane 20 and extend upwardly along the inner sidesof the bores in the gasket 22 and the bores 26 in the upper plate 24.Also included in the well assembly 12 is a cover 34 which is preferablymade of a clear, plastic material and is receivable on the Teflon plate24 so that it covers the wells 13. The cover 34 has a centrally disposedtubular sleeve 36 formed therein which extends downwardly to the plate24; and a passage 38 which is substantially aligned with the sleeve 36extends downwardly through the upper plate 24, the gasket 22, and themembrane 20 to the stainless steel plate 18.

The stimulator pin assembly 14 comprises a base 40, a stepping motor 42,a pin plate assembly generally indicated at 44, a lower limit switchassembly 46, and an upper limit switch assembly 48. The base 40comprises a substantially flat bottom base plate 50 having a pair ofspaced, upwardly extending sidewalls 52 thereon, support plate 54 whichis received and secured on the upper ends of the sidewalls 52, and anupper sidewall frame 56 on the support plate 54. The well assembly 12 isreceived on the upper ends of the upper sidewall frame 56, and it issecured to the support plate 54 with threaded rods 58 having nuts 60thereon. The stepping motor 42 preferably comprises a 4-phase, linearactuator stepping motor, such as a Model LAS motor made by HurstManufacturing Corp. of Princeton Ind., and it is mounted on the supportplate 54. The motor 42 includes a threaded shaft 62 which is connectedto the pin plate assembly 44, and it is actuatable to linearly move theshaft 62 in minute-stepped increments in order to raise or lower the pinplate assembly 44 in a substantially continuous motion and/or arepetitive motion. The pin plate assembly 44 includes a main plate 64which is attached to the shaft 62, and a plurality of pins 66 whichextend upwardly from the main plate 64. The main plate 64 is attached tothe shaft 62, and it is contained within the sidewalls 56 to prevent itfrom rotating as the shaft 62 is linearly moved, although it is freelymovable upwardly therewith. The pins 66 have blunt upper ends, and theyare positioned on the plate 64 so that they pass through the aperturesin the plate 18, and they are substantially aligned with the centers ofthe wells 13 as illustrated. Accordingly, the pins 66 are engageablewith the membrane 20 in the wells 13 to flex or expand the membrane 20upwardly in the center portions of the wells 13. The lower limit switch46 is of conventional construction, and it includes an arm 68 which isengageable with the lower end of the shaft 62 so that it defines a lowerlimit position for the shaft 62. The upper limit switch assembly 48includes an arm assembly 70 which is mounted on the base 40 and a limitswitch element 72 on the arm assembly 70. The arm assembly 70 ispivotable outwardly to enable the cover 34 to be removed, and it is alsopositionable in the manner illustrated in FIGS. 1 and 2, wherein thelimit switch element 72 is substantially aligned with the sleeve 36. Thelimit switch assembly 48 also includes a shaft 74 which extendsdownwardly through the sleeve 36 and the passage 38 and rests on themain pin plate 64 so that it moves upwardly with the main pin plate 64and is engageable with the switch element 72 for defining an upper limitposition for the pin plate 64.

The controller 16 preferably comprises a computer, such as an Apple IIecomputer, which is interfaced with the stepping motor 42 through adigital interface card (DIO9, Interactive Structures, Inc., MalvernPenna.) and optical data links (UI16 Isolated Power Interface Systemwith 4 DC-0 Output Modules, Interactive Structures). The controller 16is operative for controlling the stepping motor 42 to produce variouspredetermined activity patterns, such as the pattern illustrated in FIG.5. In this connection, the controller 16 is preferably operated tocontrol the motor 42 for gradually and substantially continuouslystretching the membrane 20 by advancing the pin plate assembly 44upwardly in minute-stepped increments over a prolonged period of timeand for also periodically repetitively stretching and releasing themembrane 20 by reciprocally moving the pin plate assembly 44 upwardlyand downwardly at predetermined intervals during the same period oftime.

For use and operation of the apparatus 10, tissue specimens 76illustrated in FIG. 4 are placed on the membrane 20 in the wells 13. Thetissue specimens may comprise various types of living tissue, such asvarious types of nerve or muscle tissue, and they are prepared andapplied to the membrane 20 in accordance with conventional laboratorytechniques. After the tissue specimens 76 have been plated on themembrane 20, the apparatus 10 is placed in a humidified incubator tomaintain the tissue specimens 76 in a life support environment, and thetissue specimens 76 are allowed to attach to the membrane 20 and/orsupport ring 30. A suitable nutrient medium is periodically supplied tothe specimens 76; and the specimens 76 are maintained in a sufficientextracellular matrix to prevent damage thereto during stretching byapplying additional collagen or another suitable matrix material theretoif and when needed as determined by known techniques. In thisconnection, with some types of tissue cells, such as skeletal musclecells, it is necessary to apply a matrix, such as a collagen gel, toprovide sufficient support for the cells during stretching; although,with some other types of tissue cells, such as some nerve cells whichare capable of producing sufficient quantities of extracellular matrixmaterial, externally applied matrices are not required. In any event,after the tissue specimens 76 are attached to the membrane 20 and/orsupport ring 30, which may take up to 24 hrs in the case of isolatedtissue cells, the controller 16 is actuated to control the steppingmotor 42 to gradually and substantially continuously stretch the tissuespecimens 76 over a prolonged period of time while periodicallyrepetitively stretching and releasing the tissue specimens 76 at spacedintervals during the same period of time. With certain types of tissuespecimens, during this portion of the method the tissue specimensactually attach themselves to the rings 30 and detach themselves fromthe membrane 20 so that the rings 30 actually support the tissue. Inthis connection, it has been found that when some tissue specimensattach themselves to the rings 30, they actually become interwoven inthe wire mesh of the rings 30 so that they are capable of withstandingstretching activity without being separated from the rings 30. In anyevent, during this portion of the method, the tissue specimens arepreferably repetitively stretched and released by between 0.02% and 60%,and they are preferably simultaneously continuously stretched at a rateof between 0.005 mm/hr and 1.0 mm/hr. The stretch and release activityperiods preferably last for at least approximately 5 seconds and theyare preferably separated by a rest period of at least approximately 5seconds in duration; and this procedure is preferably carried out for atleast several days. It has been found that by following this procedure,the apparatus 10 can be effectively utilized for growing adult-liketissue which are similar to tissue grown in vivo; although, in the caseof aligned cells, the cells are generally oriented so that they extendsubstantially radially outwardly from the centers of the wells 13.

A second embodiment of the apparatus of the instant invention isillustrated in FIGS. 6-9 and generally indicated at 78. The apparatus 78comprises a base 79, an expandable well assembly 80 having a pluralityof wells generally indicated at 82 therein which is mounted on the base79, a control well assembly 84 which is also mounted on the base 79, astepping motor 86 and a controller 88. For use and operation of theapparatus 78, the controller 88 is operated to control the motor 86 forexpanding the well assembly 80 so that the wells 82 are gradually andsubstantially continuously stretched over a prolonged period of time andalso periodically repetitively stretched and released during the sameperiod of time. Accordingly, when tissue specimens 90 illustrated inFIG. 9 are applied to the wells 82 and the wells 82 are stretched, thetissue specimens 90 are subjected to an activity pattern which issimilar to that illustrated in FIG. 5 so that the cells in the tissuespecimens 90 develop into adult-like structures which are similar tocells grown in vivo.

The base 79 comprises a substantially flat metal plate; and it isoperative for supporting the expandable well assembly 80, the controlwell assembly 84 and the stepping motor 86 as illustrated most clearlyin FIG. 6. A cover 91, which is preferably made of a transparent plasticmaterial, is received on the base for covering the well assembly 80.

The expandable well assembly 80 comprises a frame 92 which is secured ina fixed position on the base 79 with a plurality of frame mounts 94. Thewell assembly 80 further comprises a pair of ball bearing roller tracks96 which extend forwardly along opposite sides of the frame 92 and amovable stage 98 which is mounted so that it can travel freely on thetracks 96. A rear spacer bar 100 is mounted in a fixed position on theframe 92, and a front spacer bar 102 is mounted on the stage 98. Aplurality of stacked rear well end bars 106 are mounted as a unit on therear spacer bar 100, and a plurality of stacked front well end bars 104are mounted as a unit on the front spacer bar 102. In this connection,the well end bars 104 and 106 are mounted in substantially parallelrelation in spaced pairs; and since the front well end bars 104 aremounted on the front spacer bar 102 which is in turn mounted on thestage 98, the front well end bars 104 are movable with the stage 98 insubstantially parallel relation to the rear well end bars 106. The wells82 are illustrated most clearly in FIGS. 8 and 9 and they comprisesubstantially rectangular, open box-like structures which are made froma resiliently expandable rubberized material, such as Silastic (DowCorning Corp. TM). The wells 82 are positioned between the front andrear well end bars 104 and 106, respectively, and they are secured tothe well end bars 104 and 106 with Teflon (DuPont TM) plates 108 andscrews 110 having nuts 112. The plates 108 are preferably ofsubstantially the same dimension and configuration as the ends of therectangular wells 82 so that the side and bottom walls of the wells 82can be stretched while nevertheless maintaining the wells 82 insubstantially rectangular configuration. The bottom surfaces of thewells 82 are preferably coated with an extracellular matrix material,such as collagen, to adapt the bottom surfaces of the wells 82 to permitthe cells of tissue specimens to attach themselves if required thereto.Support screens generally indicated at 114 having rear walls 116 aresecured in the wells 82 by the plates 108 so that the rear walls 116 arecaptured between the plates 108 and the ends walls of the wells 82, asillustrated most clearly in FIG. 9. The support screens 114 furtherinclude front walls 118 which are joined to the rear walls 16 withbottom walls 120 so that the front walls 118 extend upwardly in spacedrelation to their respective rear walls 116. A pair of the supportscreens 114 are received in each of the wells 82, and the supportscreens 114 are dimensioned so that the front walls 118 of the supportscreens 114 in each well 82 are disposed in spaced relation asillustrated in FIG. 9. The support screens 114 are preferably made of abiocompatible screening material, such as stainless steel screening, andthey preferably have mesh sizes of between 4×4 meshes/in and 250×250meshes/in, and as herein embodied, they have a mesh size of 50×50meshes/in. The support screens 114 are preferably made from wire havinga diameter of between 0.31 mm and 3.76 mm, and they preferably have meshopenings of between 0.20 mm and 2.59 mm. As herein embodied, the supportscreens 114 are made from wire having a diameter of 0.31 mm, and theyhave mesh openings of 0.20 mm.

The control well assembly 84 comprises a plurality of stacked left wellend bars 122 and a plurality of stacked right well end bars 124 whichare secured to the base 79 with mounts 126 so that the bars 122 and 124are in spaced, substantially parallel pairs and a plurality of wells 128which are secured to the well end bars 122 and 124. The wells 128 aresubstantially identical to the wells 82, and they include supportscreens 114 and Teflon plates 108. However, because the well end bars122 and 124 are mounted in spaced relation at fixed positions on thebase 79, the control well assembly 84 cannot be operated to expand thewells 128.

The motor 86 preferably comprises a Hurst model LAS stepping motor madeby the Hurst Manufacturing Corp. of Princeton Ind., and it is mounted onthe base 79 with a bracket 130. The motor 86 includes a threaded, shaft132 which is connected to the stage 98 through a nonrotatable femalethreaded sleeve 134 and a fixed shaft 136. Accordingly, when the motor86 is actuated to to linearly move the threaded shaft 132, the sleeve134 is longitudinally advanced with the threaded shaft 132 to advancethe stage 98. A limit switch 138 is mounted on the base 79 adjacent theforward end of the frame 92 with a bracket 139, and a limit switch trip140 is attached to the forward end of the stage 98. The limit switch 138is connected to the motor 86 so that it is operative for deenergizingthe motor 86 when the stage 98 has reached a predetermined limit of itsforward travel.

The controller 88 is operative for controlling the operation of themotor 86 to apply a predetermined pattern of stretch activity to thespecimens 90 in the wells 82. The controller 88 preferably comprises anApple IIe computer which is interfaced with the stepping motor 86through a digital interface card, via optical data links.

For use and operation of the apparatus 78 in accordance with the methodof the subject invention, tissue specimens are positioned on or next tothe bottom surfaces of the wells 82 and 128 between the front supportwalls 118 therein, and the tissue specimens are maintained in a lifesupport environment and periodically fed nutrients over the course ofthe method. The tissue specimens are attached or allowed to attach tothe membranes defined by the collagen-coated bottom surfaces of thewells 82 and 128 or the support walls 118. Thereafter, in the case oftissue specimens which require cellular alignment, the controller 88 isoperated to control the motor 86 to move the stage 98 and the front wellend bars 104 slowly forwardly to gradually and substantiallycontinuously stretch the tissue specimens in the wells 82 until thecells thereof are substantially aligned. In any event, thereafter, thetissue specimens are maintained in a sufficient extracellular matrix toprevent tearing of the tissue specimens during stretching. In thisregard, again, a sufficient matrix may occur naturally for some types ofcells, but alternatively a sufficient matrix may be achieved by applyingan effective amount of a known matrix material, such as a collagen, tothe tissue specimens. Thereafter, the tissue specimens are gradually andsubstantially continuously stretched over a prolonged period of time andthey are periodically repetitively stretched and released during thesame period of time. In this connection, it has been found that in somecases at this point in the method tissue specimens which have beenapplied to the membrane begin to attach themselves to the front supportwalls 118, and they become detached from the collagen-coated bottomsurfaces of the wells 82; and after the tissue specimens have beenexposed to continuous and repetitive stretch activity for several days,the cells in the tissue specimens actually become fully attached to thefront support walls 118 and completely detached from the bottom surfacesof the wells 82. The tissue specimens are preferably stretched andreleased by between approximately 0.02% and 60% during the stretch andrelease activity periods, and they are preferably gradually andsubstantially continuously stretched at a rate of between 0.005 mm/hrand 1.0 mm/hr. Further, the activity periods preferably last for atleast 5 seconds and they are preferably separated by rest periods of atleast 5 seconds in duration during which the tissue specimens are onlysubjected to gradual, substantially continuous stretch activity.

It has been found that when tissue specimens are mechanically stimulatedin the apparatus 78 in the manner hereinabove set forth, the cellsthereof develop into three-dimensional tissues, which have theappearance of natural tissues grown in vivo. In contrast, it has beenfound that when tissue specimens are grown in the control well assembly84 they do not develop into mature-like in vivo tissues.

EXAMPLE

In a specific example of the method of the instant invention, embryonicavian skeletal muscle cells comprising pectoralis muscle cells from 11to 12 day old in ovo chick embryos were minced into pieces, prepared byconventional techniques and plated onto the collagen-coated bottom wallsof the wells 82 of the apparatus 78. The apparatus 78 had supportscreens 114 therein which were made of 0.31 mm stainless steel wire, andthey had 50×50 meshes/in with 0.2 mm mesh openings. The apparatus 78 wasthereafter maintained in a humidified 5% CO₂ incubator at 37.2° C.throughout the experiment. During the second day of the experiment thetissue specimens were fed fresh nutrient, and the controller 88 wasactuated to slowly and continuously stretch the wells 82 and the tissuespecimens therein at a rate of approximately 8 mm/24 hr. On the thirdday the specimens were again supplied fresh nutrient, and the continuousstretch activity was continued until the specimens had been stretchedfor a total of approximately 36 hrs. By the end of the third day, themuscle cells had become substantially aligned in the direction of thestretching activity, and on the fourth day the specimens were embeddedin a collagen gel extracellular matrix. On the fifth and sixth days, thetissue specimens were again supplied fresh nutrients; and on the seventhday the tissue specimens were supplied fresh nutrients, and thecontroller 88 was actuated to apply substantially continuous stretchactivity to the wells 82 at a rate of approximately 1 mm/24 hrs and tosimultaneously apply repetitive stretch activity to the wells 82,stretching and releasing the tissue specimens by 1 mm five times duringa 20-second period, and then resting the tissue specimens bydiscontinuing only the repetitive activity for a 10-second period. Thissame pattern of repetitive stretch activity was applied three times, andthen the tissue specimens were allowed to rest for a 30-minute periodduring which only continuous stretch activity was applied. The samerepetitive stretch activity pattern was then repeated throughout thecourse of the seventh day. On the eighth and ninth days, the specimenswere again supplied fresh nutrients and the repetitive and continuousstretch activity patterns were continued, and on the tenth day thespecimens were fully rested by applying no stretch activity thereto. Itwas noted that after the eleventh day, the cells of the specimens hadbecome fully attached to the support walls 118, and that they hadcompletely detached themselves from the membrane surfaces of the wells82 so that the support walls 118 were able to function as artificialtendons which applied stretch activity to the tissue specimens. From theeleventh to the eighteenth day, the tissue specimens were subjected toonly repetitive stretch activity, wherein the tissue specimens werestretched by 2 mm and relaxed five times during a 20-second period andthen allowed to rest for 10 seconds. This pattern was repeated threetimes and then the tissue specimens were allowed to rest for 30 minutes,and thereafter the entire pattern was repeated until the end of theeighteenth day.

It was found that after eighteen days the tissue specimens which hadoriginally been a monolayer of cells approximately 22 mm in length hadbeen stretched to 35.5 mm in length and that the muscle fibers in thetissue specimens were aligned and organized in three-dimensional musclefasicles. Further, these muscle fibers appeared very similar to maturein vivo muscle fibers.

It is seen therefore that the instant invention provides an effectiveapparatus and method for stimulating tissue growth in vitro. Theapparatus 10 and 78 are operative in accordance with the method forsimultaneously applying stretch activity patterns to tissue specimenswhich simulate the types of activity patterns to which in vivo tissue isexposed as a result of normal growth and periodic movement. It has beenfound that this type of mechanical stimulation causes tissue cells toform into natural and more mature tissue which closely resembles cellsdeveloped in vivo. Accordingly, it is seen that the method and apparatusof the instant invention represent significant advancements in thebiological art which have substantial medical and scientificsignificance.

While there is shown and described herein certain specific structureembodying the invention, it will be manifest to those skilled in the artthat various modifications and rearrangements of the parts may be madewithout departing from the spirit and scope of the underlying inventiveconcept and that the same is not limited to the particular forms hereinshown and described except insofar as indicated by the scope of theappended claims.

What is claimed is:
 1. A method of growing a tissue specimen in vitro, wherein said tissue specimen contains cells which are substantially aligned or require no alignment to simulate in vivo tissue, and said specimen is supported by an expandable support member so that it is expandable therewith comprising the steps of:(a) maintaining said tissue specimen in a life support environment over the course of said method; (b) providing sufficient nutrients to sustain said tissue specimen over the course of said method; (c) maintaining the cells of said tissue specimen in an extracellular matrix sufficient to permit stretching said tissue specimen without causing damage thereto over the course of said method; and (d) gradually and substantially continuously stretching said support member to stretch said tissue specimen by at least approximately 0.5% over the course of a 24 hr period while periodically repetitively stretching and releasing said support member to stretch and release said tissue specimen by at least approximately 0.01% for a total of at least 60 seconds during the course of said 24 hr period.
 2. In the method of claim 1, said tissue specimen being stretched and released by between 0.01% and 60% during said stretch and release activity.
 3. In the method of claim 1, said tissue specimen being gradually and substantially continuously stretched at a rate of between 0.005 mm/hr and 1.0 mm/hr.
 4. In the method of claim 1, said support member comprising a resilient membrane having a pair of spaced upwardly extending support walls thereon, said support walls being made of a biocompatible material and having opposed support surfaces thereon which have sufficiently roughened textures to permit attachment of said tissue specimen thereto, said support member and tissue specimen being stretched by moving said support walls apart.
 5. In the method of claim 1, said tissue specimen being repetitively stretched and released during activity periods of at least five seconds in duration.
 6. In the method of claim 5, said activity periods being separated by rest periods of at least five seconds in duration during which said tissue specimen is subjected to said gradual, substantially continuous stretch activity but no significant stretch and release activity.
 7. A method of growing a tissue specimen wherein the tissue specimen contains cells requiring alignment to simulate in vivo tissue comprising:(a) supporting said tissue specimen with an expandable support member, said support member being adapted to permit attachment of said tissue specimen thereto; (b) maintaining said tissue specimen in a life support environment over the course of said method; (c) providing sufficient nutrients to sustain said tissue specimen over the course of said method; (d) maintaining said tissue specimen in an extra-cellular matrix sufficient to permit stretching of said tissue specimen without causing damage thereto over the course of said method; (e) effecting attachment of said tissue specimen to said support member; (f) gradually and substantially continuously stretching said support member to stretch said tissue specimen until the cells thereof are substantially aligned in the direction of stretching movement; (g) allowing said tissue specimen to rest for at least 24 hours; and (h) gradually and substantially continuously stretching said support member to stretch said tissue specimen by at least approximatley 0.5% over the course of a 24 hr period while periodically repetitively stretching and releasing said support member to stretch and release said tissue specimen by at least approximately 0.01% for at least a total of 60 seconds during the course of said 24 hr period.
 8. In the method of claim 7, said tissue specimen being stretched and released by between 0.01% and 60% during said stretch and release activity.
 9. In the method of claim 7, said support member and said tissue specimen being gradually stretched at a rate of between 0.005 mm/hr and 1.0 mm/hr.
 10. In the method of claim 7, said support member including a membrane and a pair of spaced upwardly extending support walls on said membrane, said support walls being made of a biocompatible material and having opposed support surfaces thereon which have sufficiently roughened textures to permit attachment of said tissue specimen thereto.
 11. In the method of claim 7, said tissue specimen being repetitively stretched and released during activity periods of at least five seconds duration.
 12. In the method of claim 11, said activity periods being separated by rest periods of at least five seconds duration during which said tissue specimen is subjected to said gradual and substantially continuous stretch activity but not significant stretch and release activity. 